The interaction of free radical metabolites with DNA has been a major area of interest and speculation, but previous electron spin resonance (ESR) investigations of this area have been very limited. The reaction of the hydroxyl radical, generated by a Fenton system, with pyrimidine deoxyribonucleotides was investigated using the ESR technique of spin trapping. The spin trap t-nitrosobutane was employed to trap secondary radicals formed by the reaction of the hydroxyl radical with these nucleotides. The results presented here show the hydroxyl radical attack on thymidine, 2-deoxycytidine 5-monophosphate and 2-deoxyuridine 5-monophosphate produced nucleotide-derived free radicals. The results indicate that .OH radical attack occurs predominantly at the carbon-carbon double bond of the pyrimidine base. The ESR studies showed a good correlation with previous work produced by authors who used x- or gamma-ray irradiation to generate the hydroxyl radical. A thiobarbituric acid assay was also used to monitor the damage produced to the nucleotides by the Fenton system. These results showed qualitative agreement with the spin trapping studies.